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. 2020 May 8;3:224. doi: 10.1038/s42003-020-0959-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 7 — a Luminescence intensities based on the AlphaScreen assay to assess the interactions between biotinylated (Bio)-proteins for AtHAK1 (AH1) or AtPBL27 (PBL) and FLAG-conjugated proteins for Escherichia coli dihydrofolate reductase (DR) serving as control, AtCERK1 (AC1), and AtHAK1 proteins. Recombinant proteins synthesized using the cell-free system are presented in Supplementary Fig. 12. All the individual data points are shown with the means and standard errors (n = 3). Means indicated by different small letters are significantly different among the respective sets of data, based on a one-way ANOVA with post hoc Tukey’s HSD (P < 0.05). b AtHAK1 fused to the N-terminal fragment of Venus (nVenus) and AtHAK1 or AtPBL27 fused to the C-terminal fragment of Venus (cVenus) were co-expressed in Nicotiana benthamiana leaf cells. c FLAG-tagged AtHAK1 (AtHAK1-FLAG) and HA-tagged AtHAK1 (AtHAK1-3HA), AtHAK1-FLAG, AtHAK1-FLAG and HA-tagged AtPBL27 (AtPBL27-3HA) or AtPBL27-3HA were expressed in Nicotiana benthamiana leaf cells. Total proteins extracted from the leaves were immunoprecipitated using anti-FLAG-tag magnetic beads, subjected to SDS-PAGE, and probed with anti-HA or anti-FLAG antibody as a primary antibody. Arrowheads indicate the predicted, tagged AtHAK1 or AtPBL27 signals. Source data is presented in Supplementary Fig. 13.